Rocaglates as Antivirals: Comparing the Effects on Viral Resistance, Anti-Coronaviral Activity, RNA-Clamping on eIF4A and Immune Cell Toxicity

Abstract

Rocaglates are potent broad-spectrum antiviral compounds with a promising safety profile. They inhibit viral protein synthesis for different RNA viruses by clamping the 5′-UTRs of mRNAs onto the surface of the RNA helicase eIF4A. Apart from the natural rocaglate silvestrol, synthetic rocaglates like zotatifin or CR-1-31-B have been developed. Here, we compared the effects of rocaglates on viral 5′-UTR-mediated reporter gene expression and binding to an eIF4A-polypurine complex. Furthermore, we analyzed the cytotoxicity of rocaglates on several human immune cells and compared their antiviral activities in coronavirus-infected cells. Finally, the potential for developing viral resistance was evaluated by passaging human coronavirus 229E (HCoV-229E) in the presence of increasing concentrations of rocaglates in MRC-5 cells. Importantly, no decrease in rocaglate-sensitivity was observed, suggesting that virus escape mutants are unlikely to emerge if the host factor eIF4A is targeted. In summary, all three rocaglates are promising antivirals with differences in cytotoxicity against human immune cells, RNA-clamping efficiency, and antiviral activity. In detail, zotatifin showed reduced RNA-clamping efficiency and antiviral activity compared to silvestrol and CR-1-31-B, but was less cytotoxic for immune cells. Our results underline the potential of rocaglates as broad-spectrum antivirals with no indications for the emergence of escape mutations in HCoV-229E.

Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays

“…Finally, crystallographic data supports the requirement for polypurine RNA in the rocaglate binding site of eIF4A1 (33); Chen and coworkers use a covalent RocA probe and in vitro FP assays to show that this purine specificity extends to DDX3X (28). Informed by this body of knowledge, we used lysate to probe the effects of additives that enable RNA clamping in vitro (16, 25, 28–30).…”

“…Interest in their pharmacology was first stimulated by studies of crude Aglaia extracts showing anti-neoplastic potential in cellular and murine models of cancer (13). After the prototypical rocaglates rocaglamide (RocA) and silvestrol were found to drive this activity (14,15), investigations in models of viral, protozoan, and bacterial infection broadened the utility of these compounds to include infectious disease applications (16)(17)(18)(19)(20)(21)(22)(23)(24). Our group and others have since expanded the rocaglate family to include hundreds of derivatives and novel subclasses, each with demonstrated activity across a range of disease models (3,6,12,25); representative structures of natural and synthetic rocaglates used in this study are provided in Figure 1.…”

In Vitro Safety, Off-Target and Bioavailability Profile of the Antiviral Compound Silvestrol

“…For silvestrol, a CC 50 of 9.42 nM in the lung cancer cell line A549 [5], of 0.7 nM in the colon cancer cell line HT29 [17], of 30 nM in the hepatocellular carcinoma cell line Huh-7 [6] and of 5 nM in the embryonic kidney cell line HeLa [6] was observed, whereas in the lung fibroblast cell line MRC-5, no cytotoxicity up to 50 µM was detected [5]. In primary human immune cells, a CC 50 value of 29 nM in primary monocytes, of 46 nM in primary M1 macrophages and no cytotoxic effects up to 100 nM in primary M2 macrophages, T cells and primary dendritic cells were observed [9]. Cell viability was determined with a formazan-based assay in the colon cancer cell line Caco-2, in the lung cancer cell line Calu-3, in the liver cell line HepG2, in the embryonal kidney cell line HEK293T and in the kidney cancer cell line Caki-2.…”

“…Several viruses need the ATP-dependent DEAD-box RNA helicase elF4A for the translation of their mRNAs; therefore, the inhibition of this host enzyme by silvestrol is an interesting antiviral approach [7,8]. The advantages of targeting host factors include a decreased risk of escape mutations by the virus [9,10]. The disadvantages of such a strategy are possible pleiotropic side effects [11].…”

Comparing the Effects of Rocaglates on Energy Metabolism and Immune Modulation on Cells of the Human Immune System

“…Targeting the RNA helicase elF4A to unwind stable RNA secondary structures during translation initiation [ 3 , 4 ] seems to be a promising approach for the treatment of viral infections [ 5 , 6 ] since several highly pathogenic viruses rely on this host factor for the translation of their mRNAs. The group of specific elF4A helicase inhibitors comprises, among others, rocaglates like silvestrol, zotatifin and CR-31-B (−) [ 7 , 8 , 9 ].…”